| Haemophilus parasuis (HPS) is a commensal organism in the upper respiratory tract of conventional pig which it is belonging to the the Pasteurella Division Haemophilus genus. Under appropriate conditions, it can invade and cause severe systemic disease characterized by fibrinous polyserositis, meningitis and arthritis. HPS was a worldwide distribution and has become one of the global swine industry most affected bacterial sexually transmitted diseases, in addition the incidence and mortality also showed a significant upward trend. The study reported here focused on the etiology, epidemiology and diagnostic methods of HPS, and development of indirect haemagglutination test which also provided very important materials for HPS research and contributed for preventing and control of HPS in this area.The main research included as below:1. In this study, a strain of G-coccobacillus was isolated from pigs infected with Haemophilus parasuis. The bacteria were confirmed be5serotype of HPS by microscopic examination, biochemical assay, drug sensitivity test, PCR and serological identification. The HPS isolates needs special high nutritional condition, and can not grow in the common nutrient agar plate, but erupt ganoid, humid, subtransparent, edge concinnous, little intumescent center, needle tip to medium sized coenobium in the TSA plate containing NAD. Morphology of the bacteria was multiple, and exhibited filament, short rod and even ball in shape. It is consistent with the basic characteristics of HPS According to the HPS strains isolated from16SrRNA sequence design primer pairs PCR amplification, further confirmation of the isolated strains of Haemophilus parasuis.2. The isolates were CAMP test presents positive reaction. The use of micro-biochemical tube biochemical tests to get it is in line with the biochemical character of HPS that can ferment glucose, maltose, sucrose, lactose, fructose and nitrate reductase. Drug sensitivity tests revealed that the HPS was sensitive to Cefotaxime, Vanguard ADM IV and Sulbactam etc, moderate sensitive to Furazolidone and Vanguard ADM V etc, but resistant to Gentamicin, Cotrimoxazole, Penicillin, etc. The isolates strain were made of saline bacterial suspension,detection was carried out according to the method described in the directions of standard serum kit of HPS. The result showed that isolated strain has stronger coagulation reaction with the positive serum of Hps serotype5, in which white laminar coagulate was formed, but no coagulation reaction with other standard positive serum samples and normal saline. Show that the isolates is5serotype of HPS.3. Through the study of the growth curve of the isolates, show that the bacteria in TSB liquid medium was created the solution pH value decreased quickly. In the36hours of observation, the PH value reached a minimum at cultured for20h later, and the PH value is only about6.0. which is it showed, the HPS acid strong production in the training process. By using the spectrophotometer determination of OD600medium value. The result showed that the bacteria in TSB liquid medium around2h later, the OD600began to gradually decline, then it reached a maximum in between of18-24h. In the subsequent time, OD600began to gradually decline.4. This study was based on the indirect ELISA method to detect the HPS antibodies with some optimized and improved. First, we were prepared for Anti-Swine HPS positive-serum, by utilizing the isolated strain bacteria of treated with ultrasonication as the antigen. Secondly, this assay was optimized for an antigen coating concentration of1:100and a serum dilution of1:160by chessboard titration tests. The optimal dilution of HRP-Rabbit Anti-Swine IgG was1:5000. Finally, The method of sensitivity, specificity and reproducibility experiments show that, the detection sensitivity of the target antibody in indirect ELISA method based on this isolate can reach over1:10000, and this detection method has high specificity and high repeatability; It is determined as negative when detecting positive-serum of SP, E.coli, APP,ect. Which is indicating that it has good specificity; And experiments also demonstrated that the HPS antibody positive detection rate was also higher... |
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